The human corneal endothelium is the most important monolayer of cells in the cornea. It plays a significant role in maintaining corneal transparency by regulating the leaky barriers between the aqueous humor and corneal stroma. The corneal endothelium cell layer is derived from neural crest cells during the first 16 weeks of gestation. Human corneal endothelial cells (hCENC), however, have very limited proliferative capacity in vivo and the density of hCENC in the cornea shows an inverse relationship with age. Damage to the corneal endothelium layer by accidental or surgical corneal trauma and diseases such as Fuchs' dystrophy often result in corneal edema and corneal blindness. Currently, cornea transplantation is the main treatment option for irreversible damages on the corneal endothelium. However, the success of the treatment is limited by the scarcity of transplant-grade corneas from suitable donors. Hence, fresh cadaveric corneas are often used for isolation and expansion of hCENC, which have been regarded as a potential source of cells for replacing damaged corneal endothelium.
The anterior chamber of the eye is an immune-privileged site. This allows the acceptance of allografts with lower risks of transplantation rejections. The viability of using tissue-engineered hCENC constructs together with Descement stripping and automated endothelial keratoplasty (DSAEK) technique in rabbit model has been described in the art, whereby cultured hCENC were seeded on sheets of collagen and transplanted into rabbits. These sheets showed similar cell morphology as hCENC. Since then, hCENCs have been successfully transplanted into animal models and demonstrated its therapeutic efficiency for clinical therapy. Recently, the derivation of corneal endothelial-like cells from rat neural crest cells had been described. This opens the possibility of deriving hCENC from other cell sources such as hPSCs. One of the unique features of hPSCs is its ability to self-renew and expand indefinitely. Hence, hPSCs are a very attractive surrogate cell source for generating hCENC. However, directed differentiation of hPSCs is often not an efficient process, hence the ability to enrich for the cells of interest will be necessary. Furthermore, the lack of characterization tools has so far deterred the use of cultured hCENC for transplantation.
In another point, cultured hCENCs are characterized, i.e. visually, predominantly by their ‘cobblestone-like’ morphological appearance. Immunostaining with zonula occludins-1 (ZO-1) and sodium potassium ATPase (Na+K+ ATPase) have also been used frequently as markers for characterization of these cells. However, these markers are not hCENC-specific and are found ubiquitously expressed in many other cell types. Therefore, both ZO-1 and Na+K+ ATPase are not ideal markers for cell isolation and enrichment. Hence, there is a need for improved methods for specifically identifying and isolating hCENCs.